Antigen 60 (A60), a member of the thermostable macromolecular antigen family (TMA) and main component of old tuberculin and purified protein derivative (PPD), has been purified from the cytoplasm of Mycobacterium bovis BCG; its structure and metabolism have already been described. In the present paper, the action of A60 on humoral immunity has been analysed by an ELISA type immunoassay, and that on cellular immunity by the mouse footpad swelling test. Injection of very low A60 doses into unprimed mice produced an undetectable level of anti-A60 antibodies; the effect of a booster inoculation was not appreciable in the absence of incomplete Freund's adjuvant, but was evident when the latter was added. Higher doses of the antigen produced an appreciable primary response, and a sharp and long-lasting secondary response, which had a 10-fold higher intensity in the presence of incomplete adjuvant. No detectable delayed hypersensitivity reactions were observed in unprimed mice after footpad injection of A60, whereas clear responses were elicited in primed mice. This effect was more pronounced when the footpad was injected after a secondary response than after a primary response, and it was invariably magnified by incomplete adjuvant. It is concluded that A60 is a powerful immunogen, which is able to induce primary and secondary responses and delayed hypersensitivity reactions, effects that are adjuvant-modulated and develop concurrently.
Antigen A60 has been purified from the cytoplasm of Mycobacterium bovis BCG, and its composition has been determined: it has proved to be able to elicit immune reactions of both humoral and cellular type. Inoculation of A60 into the footpad of mice previously sensitized with the same antigen, or with whole mycobacterial cells produced a footpad swelling showing a peak at 24 h. Similar delayed hypersensitivity reactions were induced in sensitized guinea-pigs by subcutaneous injection of an A60 dose of 0.01 micrograms (minimal revealing dose). A quantity thousandfold higher (15 micrograms A60) was unable to induce in unsensitized guinea pigs the mounting of a cellular immunisation against A60, as shown by negative cutaneous testings 1 month later. Our results show that A60 preparations satisfied the requirements of the European Pharmacopoeia Commission and met the WHO recommendations for new tuberculins. Handicaps of old tuberculin and PPD (heterogeneous mixtures titrated biologically and unstable in solution) can be overcome by A60 preparations (a single antigen spectrophoretically measurable and stable).
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